In addition to their established role as cytotoxic effector cells, previous studies proposed that NK cells may regulate B cell responses to antigens. Both enhancement and suppression of Ig synthesis by NK cells have been reported. Our previous studies have also demonstrated that NK augmentation is mediated on both IgG and IgM responses. The results described above strongly suggest that components of the innate immune system (in this case NK cells) can provide an amplification/instructive role for the development of humoral immune adaptive responses. In order to investigate whether these immunoregulatory effects are physiological, we have developed a mouse model which recapitulates the findings with human cells. Using highly purified splenic B, T, and liver-associated NK cells, we have been able to show that co-culture of NK and B cells resulted in increased production of IgG and IgM in vitro, while co-culture of B cells with purified T cells had little or no effect. Specifically, co-culture of B cells with liver NK cells from either untreated mice or mice treated in vivo with IL-2 resulted in increased murine Ig (IgG and IgM) production whereas T cells failed to produce this enhancing effect. Therefore, the successful development of a mouse model which recapitulates the human NK/B effects will allow us to examine the mechanisms for this effect and extrapolate those findings to physiological primary and secondary antibody responses after challenge with different antigens. These studies will allow us to determine the role, if any, that Ly49 family members on NK cells play in the development of humoral immune responses, and may provide new insight into more effective generation of beneficial antibody responses or the role of Ly49s in the regulation of pathologic humoral immune responses. Natural killer cells are the major effectors of acute rejection of incompatible bone marrow cell (BMC) grafts in lethally irradiated mice. The immunogenetics of BMC rejection are largely controlled by the coexpression (or not) of inhibitory and stimulatory Ly-49 receptors whose ligands are class I major histocompatibility complex (MHC) molecules. The majority of the BMC rejection studies involved low numbers of BMCs that were resisted by host NK cells. Our collaborators and we have shown that larger numbers of BMCs were given in which rejection was not detected and the role of different Ly-49 NK subsets not presumably involved in the rejection of a particular BMC haplotype was examined. Surprisingly, the data show that the removal of NK cell subsets expressing Ly-49 inhibitory receptors for donor class I antigens, which would be predicted to have no effect on the BMC rejection capability, resulted in the marked rejection of BMCs where no resistance was normally seen. These results extend the "missing self" hypothesis to suggest that NK Ly-49 inhibitory receptors can both inhibit activation and killing by those cells, but also can in some way influence the function of NK cells that do not express the inhibitory receptor in a cell-cell interaction. This suggests that caution must be exercised before removal of host NK cell subset is applied clinically because enhanced BMC rejection may result. Thus the balance of Ly-49 NK subsets may also affect other in vivo activities of these cells. Our collaborators and we have previously shown that inhibitory receptor blockade of syngeneic NK cells was an effective means of ex vivo purging of leukemia-contaminated bone marrow, and transplantation of mice with the purged BMC resulted in long-term, relapse-free survival. We have extended the investigation to assess the anti-tumor effects mediated by NK cells H2-allogeneic to tumor cells. We demonstrate that various tumor cell lines are more susceptible to lysis by H2-allogeneic versus syngeneic NK cells in vitro even though comparable percentages of Ly-49 NK cells were present. Use of allogeneic NK cells to purge leukemia contaminating BMC prior to transplantation resulted in higher proportion of mice with long-term survival compared to the use of syngeneic NK cells. Allogeneic NK cells did not suppress hematopoietic reconstitution as measured CFU-GM, CBC, and donor chimerism at various days after transplantation. Inhibitory receptor blockade of allogeneic NK cells also significantly increased these anti-tumor effects at lower NK:tumor ratios compared to that of syngeneic NK cells. These results demonstrate that H2-allogeneic NK cells mediate more potent anti-tumor effects than syngeneic NK cells without adverse hematologic effects and thus may be useful in cancer therapy.